The goal of the proposed research is to develop a three-dimensional (3D) vascular bed from natural tissues, which can ultimately be used in scaffolds for tissue engineering applications requiring immediate vascularization or which can be used as stand-alone grafts for necrotic tissues in the body. To create the vascular construct, a decellularization procedure, which the PI has previously developed to accurately preserve the intricate micro-architecture of peripheral nerve, will be used to remove the immunogenic cellular components from highly vascularized lung tissue and to simultaneously preserve the microvessel extracellular matrix. This acellular vascular bed will be subsequently re-endothelialized with human mesenchymal stem cells (hMSCs) that have been trans differentiated into an endothelial cell phenotype, based on previous work of the Co-I. Decellularized tissues offer excellent clinical opportunities; in fact, many examples of current regenerative therapies utilize natural, acellular tissues (e.g., SIS products from Cook Biotech, AlloDerm from LifeCell, and Avance from AxoGen; note: the Avance nerve graft is based on the PI's decellularization processes for nerve). In addition, MSCs offer great potential for clinical translational because they are immune-privileged or they can be isolated from the patient and expanded ex vivo. In Specific Aim 1, highly vascularized lung tissue will be decellularized using the PI's previous decellularization protocol and subsequently characterized for matrix preservation, cellular removal, and in vivo immune response. Particular focus will be on preserving the 3D vascular interconnected network of large vessels and capillaries. The decellularization method has been effective in maintaining basal laminae of 5-10 microns diameter in nerve, supporting the hypothesis that this method can maintain capillary networks composed of the same matrix proteins. In Specific Aim 2, the decellularized vascular bed will be re-endothelialized by injecting transdifferentiated human MSCs into the vascular axis of the tissue. Human MSCs will be transdifferentiated in a poly(ethylene glycol) (PEG) crosslinked fibrin matrix (PEGylated fibrin) towards endothelial lineages, as performed previously by the Co-I. The ability of these cells to expand and form lumen inside the 3D acellular vascular construct will be evaluated. Once seeded, these transdifferentiated hMSCs will be subjected to pulsatile flow, to precondition the cells for physiological stresses that are found in the native vascular system. The vascular constructs developed in this proposal could be used to promote vascularization and regeneration of tissues in critically-sized defects (>100 microns) in a multitude of tissue types, as well as be used as a model system to investigate the properties of transdifferentiated hMSCs. PUBLIC HEALTH RELEVANCE: As the organ wait list continues to exceed the number of donors each year, the need for alternative therapies to transplantation is becoming increasingly important. Yet, researchers have demonstrated that a vascular connection is necessary in order to maintain viability in tissues beyond 100-200 microns thick. To date, there is no effective manner in which to recreate this vascular connection. The goal of our proposed research is to develop three-dimensional vascular beds from natural tissues, which can ultimately be used in scaffolds for tissue engineering applications requiring immediate vascularization or which can be used as stand-alone grafts for necrotic tissues in the body. To create the vascular construct we will use a decellularization procedure, which we have previously developed to accurately preserve the intricate micro-architecture of peripheral nerve, to remove the immunogenic cellular components from highly vascularized lung tissue and to simultaneously preserve the microvessel extracellular matrix. The vascular bed will be subsequently re- endothelialized with human mesenchymal stem cells (MSCs) that have been transdifferentiated into an endothelial cell phenotype. MSCs offer great potential for clinical translational because they are immune- privileged or they can be isolated from the patient. In addition, decellularized tissues offer clinical opportunities; in fact, most examples of current scaffold-based regenerative therapies utilize natural, acellular tissues (e.g., SIS products from Cook Biotech, AlloDerm from LifeCell, and Avance from AxoGen; note: the Avance nerve graft is based on the PI's decellularization processes for nerve tissue).